Reformulating lipid nanoparticles for organ-targeted mRNA accumulation and translation

Fully targeted mRNA therapeutics necessitate simultaneous organ-specific accumulation and effective translation. Despite some progress, delivery systems are still unable to fully achieve this. Here, we reformulate lipid nanoparticles (LNPs) through adjustments in lipid material structures and compositions to systematically achieve the pulmonary and hepatic (respectively) targeted mRNA distribution and expression. A combinatorial library of degradable-core based ionizable cationic lipids is designed, following by optimisation of LNP compositions. Contrary to current LNP paradigms, our findings demonstrate that cholesterol and phospholipid are dispensable for LNP functionality. Specifically, cholesterol-removal addresses the persistent challenge of preventing nanoparticle accumulation in hepatic tissues. By modulating and simplifying intrinsic LNP components, concurrent mRNA accumulation and translation is achieved in the lung and liver, respectively. This targeting strategy is applicable to existing LNP systems with potential to expand the progress of precise mRNA therapy for diverse diseases.


Lipid fusion by FRET analysis
The fusion of LNPs with endosomal mimicking liposomes was assessed by the FRET assay 1 .The FRET probes, NBD-PE and Rho-PE, were formulated in the endosome-mimicking nanoparticles, and the NBD fluorescence was diminished due to the FRET to rhodamine.When the lipid fusion occurred, the increased distance between the two probes would lead to an NBD signal increase.Endosomal mimics were prepared by mixing DOPC/DOPS/DOPE/Rho-PE/NBD-PE (molar ratio of 25/25/48/1/1) in chloroform.The thin lipid films were obtained by rotary evaporation, followed by vacuum drying for 2 h to remove the solvents.Subsequently, 1× PBS buffer (pH 7.4) was added to the dried lipid films and sonicated for 30 min, giving a total lipid concentration of 1 mM.nAcx-Cm LNPs were prepared with a total lipid concentration of 1 mM, and were formulated at nAcx-Cm/DOPE/cholesterol/DMG-PEG2000 molar ratio of 15/20/25/2.Black 96-well plate wells were spiked with 100 μL of 1× PBS buffer at pH 5.5 and pH 7.4, respectively.Afterwards, 1 μL of anionic endosomal mimicking liposomes and 10 μL of nAcx-Cm LNPs were added into the wells.Post incubation at 37 °C for 5 min, the fluorescence (F) was measured at Ex/Em of 465/520 nm on a microplate reader.Anionic endosomal liposomes in 1× PBS were set as the negative control (Fmin).The lipid mixtures containing FRET probes and Triton X-100 (1 wt.%) were set as the positive control (Fmax).Lipid fusion rates (%) were calculated as (F-Fmin)/(Fmax-Fmin) ×100%.

Endosomal escape and cellular uptake assay
Endosomal escape and cellular uptake of LNPs were determined by the confocal imaging.IGROV1 cells were inoculated into glass-bottomed/confocal culture dishes at a density of 200,000 cells/well.After 24 h, 1 mL of fresh medium (10% FBS) was added and the cells were treated with LNPs containing 500 ng Cy5-mRNA.LNPs were formulated with nAcx-Cm /DOPE/Cholesterol/DMG-PEG2000 molar ratio of 15/20/25/2. 4 h later, cells were washed three times with 1× PBS.The cells were stained with Lysotracker Green DND26 (1/8000 dilution) for 40 min at 37 °C, and then stained with Hoechst 33342 (0.1 mg/mL) for 5 min.Finally, the cells were imaged by the confocal microscopy.

LNP dissociation by FRET assay
The dissociation of nAcx-Cm LNPs was determined by mixing 6Ac1-C12 LNPs with the endosomal mimicking anionic liposomes.The DOPE-derived FRET probes NBD-PE and Rho-PE were prepared into the same LNPs.The specific formulation used 6Ac1-C12/DOPE/Cholesterol/DMG-PEG2000/NBD-PE/Rho-PE (molar ratio 15/20/25/2/0.63/0.63)lipid mixture to prepare the 6Ac1-C12 LNPs, with a total lipid concentration of 1 mM.The lipid mixture comprising DOPS/DOPC/DOPE with a molar ratio of 25/25/50 was formulated to get the endosomal mimicking anionic liposomes.Briefly, the liposomes in chloroform were subjected to rotary evaporation and vacuum drying for 2 h to obtain the lipid films.Subsequently, the dried films were then hydrated by sonication in 1× PBS buffer (pH 7.4) for 30 min to give a total lipid concentration of 10 mM.The 1× PBS buffer at pH 5.5 (100 μL/well), 1 μL of 6Ac1-C12 LNPs and 1 μL of endosomal mimicking anionic liposomes were added to the black 96 well plates.Following incubation at 37 °C for 5 min or other time intervals, the fluorescence (F) was measured on a microplate reader at Ex/Em of 465/520 nm.The negative control (Fmin) was set as 6Ac1-C12 LNPs incorporating the FRET probes in 1× PBS buffer.The positive control (Fmax) was set as 6Ac1-C12 LNPs containing FRET probes incubated with Triton X-100 solutions (1 wt.%).The dissociation of LNPs (%) was calculated as (F-Fmin)/(Fmax-Fmin) ×100%.

mRNA release assay
The mRNA release of nAcx-Cm LNPs was determined by mixing 6Ac1-C12 LNPs with endosomal mimicking anionic liposomes.The 6Ac1-C12 LNPs contained 6Ac1-C12/DOPE/Cholesterol/DMG-PEG2000 with a molar ratio 15/20/25/2 (total lipid concentration, 1 mM).The endosomal mimicking lipid mixture was formulated at DOPS/DOPC/DOPE molar ratio of 25/25/50 in chloroform.The anionic lipid films were obtained by rotary evaporation followed by vacuum drying for 2 h to remove the solvents.Afterwards, the lipid film was hydrated by sonication in 1× PBS to give a total lipid concentration of 10 mM. 1 μL of 6Ac1-C12 LNPs and 1 μL of endosomal mimicking anionic liposomes were mixed, incubating at 37 °C for 5 min or 30 min.The mRNA release was detected by Quant-iT Ribogreen RNA assay kit using the microplate reader.
pKa determination using the 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay The pKa of the LNPs was determined by TNS assay.LNPs were comprised of synthetic ionizable lipid/DOPE/cholesterol/DMG-PEG2000 (molar ratio 15/20/25/2) in the 1× PBS buffer with a total lipid concentration of 1 mM.The TNS was diluted to a 100 μM solution using milliQ water.Each well of the black 96-well plates was spiked with 100 μL of buffer solution containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM 4-morpholineethanesulfonic acid (MES), 10 mM ammonium acetate, and 130 mM sodium chloride (NaCl) with a pH range of 2.5 to 11.The LNPs were diluted to a final 10 μM total lipid concentration in the wells.The TNS solution was added to each well, making a final concentration of 2 μM.The plates were read using a microplate reader at Ex/Em of 321/445 nm.The fluorescence data was analyzed by a sigmoidal fit analysis and the pKa was determined as the pH at half of the maximum fluorescence intensity.

Hydrolysis of ionizable lipid 6Ac1-C12
Lipid hydrolysis analysis was conducted as per the literature 2 .6Ac1-C12 lipid (177.4 mg, 0.10 mmol) was added to a 50 mL round bottom flask.Next, 10 mL of 6 M HCl aqueous solution was added to give a cloudy non-homogeneous dispersion.Reaction was heated and refluxed for 24 h to obtain a clear solution.Then the mixture solution was cooled and neutralized with 4 M NaOH aqueous solution.Chloroform was utilized for extraction, and the combined organic solution was concentrated to obtain a degradable mixture of 6Ac1-C12 byproducts.The degradation mixture was analyzed by 1 H NMR.
For the isolation of mouse liver cells, differential centrifugation was employed 3 .The mice were anesthetized with isoflurane and fixed on the foam plates.The mouse liver was perfused, and then clipped and digested with collagenase IV at 37 °C for 30 min.Following termination of digestion, liver cells were passed through a 70 μm cell filter and washed with 1× PBS.Subsequently, hepatic parenchymal cells were collected by centrifugation (4 °C, 50 g, 5 min), and the cell sediment was resuspended in the washing medium and then washed with 1× PBS.The supernatants were collected and the liver nonparenchymal cells were obtained by centrifugation (4 °C, 450 g, 5 min).These cells were resuspended in the red blood cell (RBC) lysis buffer and incubated for 5 min.The RBC lysis was terminated by the addition of 1× PBS.The mixture was centrifuged, counted and then resuspended in the cell staining buffer.Antibodies for staining were added into the cell suspension and incubated on ice for 30 min in the dark.After staining, the cells were washed twice with 1× PBS and finally resuspended in 500 μL of 1× PBS.Afterwards, the cell suspensions were transferred to the flow tube and analyzed with the multicolor analytical flow cytometer (LSR Fortessa, BD Biosciences).Antibodies used here included PerCP/Cyanine5.5 anti-mouse CD45 (1/200 dilution, Biolegend, 157208), PE/Cyanine7 anti-mouse CD31 (1/200 dilution, Biolegend, 102524), FITC antimouse/ human CD11b (1/100 dilution, Biolegend, 101205), and Brilliant Violet 785 TM antimouse F4/80 (1/100 dilution, Biolegend, 123141).Sytox TM Blue Dead Cell Stain (1/2000 dilution, Thermo Fisher, S34857) was employed to differentiate the live cells.
To perform isolation of lung cells, mouse lungs were minced in plates and then transferred to the 15 mL centrifuge tubes containing collagenase I and DNase I digestion medium.The mixture was digested by shaking at 37 °C for 1 h in a constant temperature shaker.Post termination of digestion, the mixture was passed through a 70 µm cell filter and washed with 1× PBS.Pulmonary cells were collected by centrifugation (4 °C, 450 g, 5 min).Antibodies used here included PerCP/Cyanine5.5 anti-mouse CD45 (1/200 dilution, Biolegend, 157208), PE/Cyanine7 anti-mouse CD31 (1/200 dilution, Biolegend, 102524), and FITC anti-mouse CD326 (Ep-CAM) (1/100 dilution, Biolegend, 118207).Sytox TM Blue Dead Cell Stain (1/2000 dilution, Thermo Fisher, S34857) was used to differentiate the live cells.Ultimately, lung cells were analyzed using the LSRForessa machine.Data were analyzed using FLOWJO software version 10.8 (FLOWJO) Isolation of protein coronas adsorbed on LNPs in plasma 6Ac1-C12 Liver and Lung LNPs were prepared and diluted to a total lipid concentration of 5 mg/mL with 1× PBS.Different LNPs were co-incubated with mouse plasma for 60 min at 37 °C.The mixture was then centrifuged at 15,000 g for 30 min at 4 °C.The supernatant was removed and the pellet was washed with 1× PBS.The washing process was conducted for a total of 3 times.The obtained plasma protein samples were stored at -20 °C for further analysis.The same procedure was conducted with plasma/PBS to certify the absence of plasma precipitation by centrifugation.Supplementary Fig. 29 | The FACS gating strategy for analysis of tdTomato expression in lung cells.Sytox TM Blue was used to identify live and dead cells.EpCam+ was utilized to define epithelial cells, CD45+ and CD31-was used to define immune cells, and CD45-and CD31+ were used to define endothelial cells.Gates for tdTomato+ in cell types were drawn based on the PBS injected control mice.Ai9 mice were i.v.injected with 6Ac1-C12 3-Comp Lung LNPs (Cre mRNA, 0.25 mg kg ) and tdTomato+ in given cell types was detected by flow cytometry.